Rapid and stable transformation method of agrobacterium tumefaciens-mediated sunflower

Abstract

The present disclosure provides methods of gene transformation of sunflower based on Agrobacterium-mediated gene delivery to meristem cells of fresh imbibed / germinated sunflower seeds. One embodiment of the present disclosure is a method of directly transforming a sunflower from a mature seed with a heterologous polynucleotide, comprising: generating an explant from a germinated sunflower seed by removing cotyledons and primary leaves having intact meristem and tender shoot radicles; contacting the sunflower explant comprising the cotyledon with Agrobacterium comprising a heterologous polynucleotide in an infection medium supplemented with a plant hormone using vacuum and ultrasound treatment; co-culturing the explant from a germinating sunflower plant in a culture medium lacking the plant hormone in the presence of the Agrobacterium genus; culturing the mature seed explant in a regeneration medium comprising a selective agent; and culturing the seed with the regenerated tender shoots and with the secondary roots in a culture medium lacking the selective agent.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, particularly to the rapid transformation of sunflower mediated by Agrobacterium. Relevant application information

[0002] This application claims priority to application PCT / CN2023 / 088375, filed on April 14, 2023, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0003] Sunflower (Helianthus annuus L.) is one of the major edible oilseed crops cultivated worldwide, ranking fourth in global vegetable oil production. However, due to the lack of stable and efficient conversion systems, crop / trait improvement in sunflowers is much slower than in other major crops such as maize, rice, wheat, and soybeans. To date, genetic improvement has been achieved through traditional breeding that utilizes the natural genetic diversity among sunflower varieties. This approach has considerable limitations for trait improvement.

[0004] Several public reports exist on sunflower transformation. Scltiammeijer et al. first generated transgenic sunflowers by transplanting transformed shoots onto non-transgenic rootstocks to obtain transgenic seedlings, but the efficiency was quite low (Scltiammeijer et al. (1990) Plant Cell Reports 9:55-60). Mushke et al. described the transformation of sunflower hypocotyl explants; however, this protocol was inefficient and required a long timeline (approximately 103 days) (Mushke et al. (2019) Molecular Biology Reports). Therefore, although sunflower transformation was demonstrated in the 1990s, the transformation efficiency was extremely low, and the timeline was 3 months or longer. Germplasm-independent transformation is lacking.

[0005] Therefore, a stable and efficient sunflower transformation system capable of producing high-quality transgenic events is needed to promote sunflower trait research and crop improvement. Summary of the Invention

[0006] This disclosure provides methods for gene transformation in sunflowers based on Agrobacterium-mediated gene delivery into meristematic cells of fresh imbibition / germination sunflower seeds.

[0007] One embodiment of this disclosure relates to a method for directly transforming sunflower (Helianthus annuus) seeds from mature seeds using heterologous polynucleotides, the method comprising: removing the radicle with intact meristem and shoot (shoot) The process involves generating explants from germinating sunflower seeds using one cotyledon and primary leaf of a radical; gently injuring the apical meristem region with the sharp tip of a scalpel; optionally, using vacuum and sonication to contact the sunflower explant containing one cotyledon with *Agrobacterium tumefaciens* containing heterologous polynucleotides in an infection medium supplemented with plant hormones; co-culturing the explants from germinating sunflower plants in the presence of *Agrobacterium tumefaciens* in a medium; culturing mature seed explants in a regeneration medium containing selectants (e.g., antibiotics such as spectinomycin and bensulfuron-methyl); culturing seeds with regenerated shoots, with or without secondary roots, in a medium lacking selectants (e.g., antibiotics such as spectinomycin and bensulfuron-methyl); and transplanting the explants into the soil under conditions allowing for further development.

[0008] In some embodiments, these methods involve germinating sunflower seeds for approximately one day, followed by the generation of explants. These methods also include modifying Agrobacterium tumefaciens to include heterologous polynucleotides (e.g., genes).

[0009] In some embodiments, contacting sunflower explants containing cotyledons with *Agrobacterium tumefaciens* containing heterologous polynucleotides in an infection medium supplemented with plant hormones is performed using vacuum and sonication. In some embodiments, vacuum and sonication improve infection efficiency. While a variety of different plant hormones can be used, in some embodiments, the plant hormones comprise TDZ and / or BAP, zeatin nucleoside. In some embodiments, about 3-20 mg / L of plant hormone is used. In some embodiments, the method includes contacting sunflowers in an infection medium containing plant hormones for about 4 hours or less.

[0010] In other embodiments, co-cultivation includes cultivation with or without light. For example, in some embodiments, co-cultivation includes cultivation under low light. The method may include co-cultivation for approximately 4-6 days.

[0011] In some embodiments, culturing mature seed explants in a regeneration medium involves inserting cotyledonary / meristematic regions into a solid medium in the presence of a selector (e.g., antibiotics such as spectinomycin and bensulfuron-methyl), covering the seeds with a soil matrix, and allowing the seeds to grow. In some embodiments, the culture lasts approximately 7–14 days. In alternative embodiments, each day of culture includes 16 hours of culture in the presence of light and 8 hours of culture in the presence of darkness. In other embodiments, the culture lasts approximately 7–14 days and includes culture in darkness for approximately the first 2–3 days.

[0012] In some embodiments, seeds with regenerated shoots, with or without secondary roots, are cultured in a medium lacking selectants (e.g., antibiotics). The culture lasts approximately 3–7 days.

[0013] In other embodiments, the explants are transplanted into the soil under conditions that allow for further development. This includes allowing the explants to grow for approximately 2–3 weeks without any selection (e.g., any selectant such as antibiotics).

[0014] The method may also include additional steps, such as characterizing the transgenic plant and / or determining the presence of heteropolynucleotides in the transgenic plant.

[0015] This disclosure also includes plants or plant parts produced by any of these methods. Additionally, this disclosure includes progeny seeds produced by hybridization of such plant with a second plant or by self-pollination of the plant. Furthermore, this disclosure includes derivatives or trade products derived from or obtained from plants or plant parts. Attached Figure Description

[0016] The foregoing overview and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. The drawings illustrate embodiments of the invention for the purpose of illustrating the invention. However, it should be understood that the invention is not limited to the precise configurations, examples, and means shown.

[0017] Figure 1 outlines the general steps of the methods currently used for conversion.

[0018] Figure 2B and Figure 2A The general steps of the method disclosed herein are outlined.

[0019] Figure 3 A diagram of explants used for infection is shown. Explants are generated by removing primary leaves and creating wounds between the apical regions.

[0020] Figure 4A and Figure 4B The explants were shown after four days of co-culturing with Agrobacterium.

[0021] Figure 5A and Figure 5B The expression of green fluorescent protein (GFP) in explants after co-culture is shown.

[0022] Figures 6A-6D The GFP expression of the explant (SF69 A) was shown 9 days after regeneration.

[0023] Figures 7A-7C The explant used for transplantation is shown.

[0024] Figure 8A and Figure 8B The small plantlets are shown 21 days after transplanting.

[0025] Figure 9 A construction diagram of carrier 24545 is shown.

[0026] Figure 10 A schematic diagram of the construction of carrier 18891 is shown.

[0027] Figures 11A-11D The detection of GFP in transgenic sunflower plants is shown. Figure 11A and Figure 11B The leaves of sunflower plants transformed with vector 24544 exhibit a positive event (successful transformation) with white and green fluorescence. Figure 11C and Figure 11D The image shows negative-negative plant leaves (i.e., unconverted) that exhibit white and green fluorescence in sunflower plants that were not successfully treated with carrier 24545.

[0028] Figures 12A-12D The detection of cyan fluorescent protein (CFP) in transgenic sunflower plants is shown. Figure 12A and Figure 12B The image shows a leaf from a sunflower plant transformed with vector 18891 that exhibits a positive event (successful transformation) with white and blue fluorescence. Figure 12C and Figure 12D The image shows negative plant leaves that exhibit white and blue fluorescence in sunflower plants that were not successfully treated with carrier 18891.

[0029] Figures 13A-13C The image shows the normal growth of the transgenic plant. Figure 13A Plants SUET210200A004A and SUET210200A005A are shown. Figure 13B The plant SUET210200A004A is shown. Figure 13C The plant SUET210200A005A is shown.

[0030] Figures 14A-14C The results show that the transgenic plant grew normally after 23 days. Figure 14AThe plant SUET210201A008A is shown after 23 days of growth. Figure 14B The plant SUET210200A004A is shown after 23 days of growth. Figure 14C The plant SUET210200A005A is shown after 23 days of growth.

[0031] Figures 15A-15C The image shows a genetically modified plant that has been grown in a greenhouse for one month. Figure 15A The image shows plant SUET210201A008A after one month of growth. Figure 15B The image shows plant SUET210200A004A after one month of growth. Figure 15C The image shows plant SUET210200A005A after one month of growth.

[0032] Figures 16A-16C The image shows a genetically modified plant that has been grown in a greenhouse for two months. Figure 16A The image shows plant SUET210201A008A after two months of growth. Figure 16B The plant SUET210200A004A is shown after two months of growth. Figure 16C The plant SUET210200A005A is shown after two months of growth.

[0033] Figures 17A-17C A comparison of transgenic plant seeds and wild-type plant seeds is shown. Figure 17A Transgenic seeds (left) and wild-type seeds (right) from plant SUET210201A008A are shown. Figure 17B Transgenic seeds (left) and wild-type seeds (right) from plant SUET210200A004A are shown. Figure 17C Transgenic seeds (left) and wild-type seeds (right) from plant SUET210200A005A are shown.

[0034] Figure 18A and Figure 18B The expression of green fluorescent protein (GFP) in transgenic sunflower seeds is shown. Figure 18A The image shows a genetically modified seed (left) and a wild-type seed (right). Figure 18B The results show that GFP expression is only visible in transgenic seeds and not in wild-type seeds.

[0035] Figure 19A and Figure 19B CFP expression in transgenic sunflower seeds is shown. Figure 19A The image shows a genetically modified seed (left) and a wild-type seed (right). Figure 19BThe results showed that CFP expression was only visible in transgenic seeds and not in wild-type seeds.

[0036] Figure 20A and Figure 20B The image shows a transgenic plant from SF81 that has grown for 21 days after transplanting.

[0037] Figure 21A and Figure 21B The image shows a genetically modified plant growing in a pot. Figure 21A Transgenic plants generated from one of the successfully transformed explants in SF-66-SP-24545-B are shown (see Table 15). Figure 21B A top view of the same plant is shown. Detailed Implementation

[0038] This disclosure provides a novel method for generating stable sunflower transformations using explants derived from imbibition seeds. These methods offer several advantages compared to current sunflower transformation methods.

[0039] The disclosed methods provide a shortened turnaround process for generating transformed sunflower plants. In some embodiments, these methods involve the following steps: (a) Explants are generated from seeds that have germinated for about one or two days by removing one cotyledon and primary leaf (with complete meristem and radicle). (b) Infection of explants was carried out by adding plant hormones such as thiabendazole (TDZ) and / or BAP, zeatin nucleoside to an infection medium containing Agrobacterium spp., and optionally by using vacuum and sonication to improve infection efficiency. (c) Incubate for 4-6 days with or without light; (d) Recovery for 9–14 days with the use of selectants (e.g., antibiotics such as spectinomycin and bensulfuron-methyl), with the first 2–3 days incubated in the dark; and (e) After recovery, the explants are transplanted into the soil for further development for 2-3 weeks.

[0040] Transgenic plants produced using the method disclosed herein exhibit normal development and agronomic performance (without premature flowering or rooting problems). Furthermore, the transformation method disclosed herein is faster than conventional methods (obtaining transgenic plants takes less than 8 weeks). definition

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this disclosure pertains.

[0042] Although the following terms are thought to be well understood by those skilled in the art, the following definitions are set forth in order to facilitate understanding of the subject matter of this disclosure.

[0043] Unless otherwise expressly stated in the context, as used herein, the singular forms “a” and “the” include plural references. Thus, for example, a reference to “cell” includes a plurality of such cells, and a reference to “protein” includes a reference to one or more proteins and their equivalents known to those skilled in the art, and so on. Unless otherwise expressly stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0044] As used in the specification and claims, the term "comprising" and its grammatical variations may include aspects of "consisting of" and "substantially consisting of". "Comprising" and its grammatical variations may also mean "comprising but not limited to".

[0045] As used in this article, the word "or" refers to any member of a particular list and also includes any combination of members of that list.

[0046] As used herein, the term “and / or” means and covers any and all possible combinations of one or more associated listed items, together with the lack of combinations when interpreted in terms of alternatives (“or”).

[0047] In this invention, a range can be expressed as from “about” one specific value and / or to “about” another specific value. When expressing a range, other aspects include from one specific value and / or to another specific value. Similarly, when a value is expressed as an approximation, it should be understood that the specific value forms another aspect by using the antecedent “about”. It should also be understood that each endpoint of a range is significantly related to, but not dependent on, another endpoint. It should also be understood that this invention discloses multiple values, each of which, in addition to the value itself, is also disclosed herein as “about” a specific value. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It should also be understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0048] The term "about" is used herein to mean approximately, roughly, around, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries to be higher than and lower than the stated value. Generally, the term "about" is used herein to modify a value to vary above and below the stated value by 20%, preferably 10%. With respect to temperature, the term "about" means ±1°C, preferably ±0.5°C. When the term "about" is used in the context of this invention (e.g., in combination with temperature or molecular weight values), an exact value (i.e., without "about") is preferred.

[0049] When applied to the polynucleotide sequences of the present invention, the term "substantially composed of" (and its grammatical variations) means a polynucleotide sequence consisting of both the described sequence (e.g., SEQ ID NO) and a total of ten or fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides at the 5' and / or 3' ends of the described sequence, such that the function of the polynucleotide is not substantially altered. The total of ten or fewer additional nucleotides includes the total number of additional nucleotides at both ends combined. When applied to the polynucleotides of the present invention, the term "substantially altered" means that the ability to express the polynucleotide sequence is increased or decreased by at least about 50% or more compared to the expression level of the polynucleotide sequence consisting of the described sequence.

[0050] As used in this article, the term "apical dominance" refers to the phenomenon where the primary shoot dominates and inhibits the growth of axillary meristems. Apical dominance is thought to be caused by auxin, which moves downwards into the axillary meristems and inhibits their growth.

[0051] As used herein, the term "axillary bud" refers to an embryonic or organogenic bud located in the leaf axil of a cotyledon or leaf. Axillary buds contain axillary meristems that can develop into shoots or flower clusters.

[0052] As used in this article, the term "axillary meristem" refers to a plant region containing stem cells located on the side of the plant stem but not at the stem tip.

[0053] As used herein, "explant" refers to a tissue, a piece of tissue, or multiple pieces of tissue derived from a plant or a part of a plant (e.g., a seed). An explant can be a part of a plant, such as an immature embryo or leaf meristem, or it can be derived from a bud, leaf, immature embryo, or any other tissue of a plant or seed.

[0054] As used herein, the term “introduction” in relation to plants means that it is accomplished in any of the following ways, including but not limited to: introgression, transgenic, clustered regular short palindromic repeat modification (CRISPR), transcription activator-like effector nuclease (TALEN) (Feng et al. 2013, Joung and Sander 2013), large-scale nuclease, or zinc finger nuclease (ZFN).

[0055] In this invention, “nucleic acid” refers to deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, it includes known analogs (e.g., peptide nucleic acids) that have the basic characteristics of natural nucleotides in hybridization with single-stranded nucleic acids in a manner similar to hybridization with naturally occurring nucleotides.

[0056] The term "variant" and its grammatical variations refer to substantially similar sequences. For nucleic acid molecules, variants include the deletion and / or addition of one or more nucleotides at one or more sites in the native nucleic acid molecule, and / or the substitution of one or more nucleotides at one or more sites in the native nucleic acid molecule.

[0057] The term "protein" refers to a polymer of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding natural amino acids, as well as to polymers containing natural amino acids.

[0058] As used in this article, "natural" nucleic acid molecules or proteins contain naturally occurring nucleotide sequences or amino acid sequences, respectively.

[0059] As used herein, the term “coding” and its grammatical variations are used to mean that a nucleic acid contains the desired information specified by using codons to guide the translation of a nucleotide sequence (e.g., a legume sequence) into a specific protein. Nucleic acids encoding proteins may contain untranslated sequences (e.g., introns) within the translated regions of the nucleic acid, or may lack such intermediate untranslated sequences (e.g., as in cDNA).

[0060] As used in this article, the term "allelic gene" refers to one of two or more distinct nucleotides or nucleotide sequences that appear at a particular locus.

[0061] A marker is "associated" with a trait when its presence indicates whether and / or to what extent the desired trait or phenotype occurs in the plant / germstone containing the marker. Similarly, a marker is "associated" with an allele when its presence indicates the presence of that allele in the plant / germstone containing the marker. For example, "a marker associated with enhanced pathogen resistance" refers to a marker whose presence or absence can be used to predict whether and / or to what extent a plant will exhibit a pathogen resistance phenotype.

[0062] As used herein, the terms “backcross” and “backcrossing” refer to the process of repeatedly backcrossing a progeny plant with one of its parents. In a backcross scheme, the “donor” parent is the parent plant that possesses the desired gene or locus to be introgressed. The “recipient” parent (used once or multiple times) or “recurrent” parent (used twice or more) is the parent plant into which the gene or locus is introgressed. For example, see Ragot, M. et al., Marker-assisted Backcrossing: A Practical Example, in Techniques et Utilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp. 45-56 (1995); and Openshaw et al., Marker-assisted Selection in Backcross Breeding, in Proceedings of the Symposium “Analysis of Molecular Marker Data”, pp. 41-53 (1994). The initial cross produces the F1 generation. The term “BC1” refers to the second use of the recurrent parent, “BC2” to the third use, and so on.

[0063] Centiliter (“cM”) is a unit of measurement for recombination frequency. One cM equals 1% of the following chance: a marker at one locus segregates from a marker at a second locus due to hybridization in a single generation.

[0064] As used herein, the term “chromosomal region defined by and including” with respect to a particular locus and / or allele means the chromosomal region defined by and encompassing the locus / allele.

[0065] As used herein, the term "cross" or "crossed" refers to the fusion of gametes via pollination to produce offspring (e.g., cells, seeds, or plants). This term encompasses both sexual hybridization (one plant being pollinated by another) and self-pollination (self-pollination, such as when pollen and ovules come from the same plant). The term "crossing" refers to the act of fusing gametes via pollination to produce offspring.

[0066] As used herein, the terms “cultivar” and “variety” refer to a group of similar plants that can be distinguished from other varieties within the same species by structural or genetic characteristics and / or phenotypes.

[0067] As used herein, the terms “desired allele,” “favorable allele,” and “target allele” are used interchangeably to refer to the allele associated with the desired trait (e.g., ASR resistance).

[0068] As used herein, the terms “inhibition,” “reduction,” etc., and their grammatical variations, refer to any reduction in the expression or function of a target gene product, including any relative reduction in expression or function, up to and including the complete elimination of the expression or function of the target gene product.

[0069] The term “enhancement” and its grammatical variations refer to improving, increasing, amplifying, replicating, raising and / or elevating to reduce one or more disease symptoms.

[0070] As used herein, the terms “increase,” “enhance,” and their grammatical variations are used to refer to any promotion or gain or increase in the expression, function, or activity of a target gene (e.g., a resistance gene) product compared to a susceptible plant, thereby providing enhanced resistance to one or more pathogens (e.g., Phakopsora) or diseases (e.g., rust). Furthermore, as used herein, the terms “lead to” or “increase,” and their grammatical variations, can refer to higher expression of a target gene product such that the level is increased by 10% or more, 50% or more, or 100% relative to cells or plants lacking the target gene or protein disclosed herein.

[0071] As used herein, the term "immune" or "immune" refers to the absence of any macroscopically visible disease symptoms. The term "partial resistance" as used herein refers to a reduction in the extent or degree of macroscopically visible lesions (without or with limited spore formation) and / or any disease symptoms and / or a delay in the progression of any disease symptoms, and may manifest, for example, as a reduction in the number of lesions or a reduction in spore formation. As used herein, with respect to rust, the term "susceptibility" or the phrase "lack of resistance" refers to the occurrence of lesions at spore formation levels equal to or higher than those observed in reference standards (e.g., Williams or Peking varieties).

[0072] As used herein, the term "resistance" refers to the absence or reduction of symptoms of one or more diseases caused by plant pathogens in a plant. Resistance may mean a reduction, minimization, or decrease in disease symptoms, such as the number of diseased plants, leaf drop, and associated yield losses, compared to disease-susceptible plants or plants that do not contain effective resistance genes that reduce symptoms of one or more diseases. Furthermore, resistance may include prevention or delay of pathogen proliferation. Generally, the term "resistance" includes both immune and partial resistance as defined above.

[0073] As used herein, the terms “enhanced pathogen resistance,” “enhanced plant pathogen resistance,” or “enhanced disease resistance” refer to an improvement, enhancement, or increase in a plant’s tolerance and / or robustness to a disease (e.g., Asian soybean rust) compared to one or more control plants (e.g., one or two parents, or plants lacking markers associated with enhanced pathogen resistance to the corresponding pathogen / disease). Enhanced disease resistance includes any mechanism that reduces the expression of symptoms indicating infection with the corresponding disease (other than whole-plant immunity or resistance).

[0074] “Plant pathogens” and its grammatical variants may be used in this article to refer to fungal pathogens of the genus Phakopsora in the class Basidiomycetes (including Phakopsora pachyrhizi and Phakopsora meibomiae).

[0075] The term "disease resistance gene" or "resistance gene" as used in this invention refers to a gene that encodes a protein that can enhance or improve the plant's defense or immune system response.

[0076] The term "orthologous" and its grammatical variants refer to genes that originate from a common ancestor and exist in different species due to speciation.

[0077] As used in this invention, the term "germstone" refers to genetic material derived from an individual (e.g., a plant), a population of individuals (e.g., a plant lineage, variety, or family), or a clone derived from a lineage, variety, species, or culture. Germstone can be part of an organism or cell, or can be isolated from an organism or cell. Germstone provides genetic material with a specific molecular composition that provides the physical basis for some or all of the genetic characteristics of an organism or cell culture.

[0078] A genetic map is a description of genetic linkages between loci on one or more chromosomes within a given species, typically presented graphically or tabularly. For each genetic map, the distance between loci is measured by the frequency of recombination between them. Recombination between loci can be detected using various biomarkers. A genetic map is the product of the mapping population, the types of biomarkers used, and the polymorphic potential of each biomarker between different populations. The order and genetic distance between loci on one genetic map may differ from those on another.

[0079] As used herein, the term "genotype" refers to the genetic makeup of an individual (or population of individuals) at one or more genetic loci, in contrast to observable and / or detectable and / or expressed traits (phenotypes). A genotype is defined by one or more alleles inherited by an individual from one or more known loci of its parents. The term genotype can be used to refer to the genetic makeup of an individual at a single locus, multiple loci, or more generally, to refer to the genetic makeup of an individual across all genes in its genome. Genotypes can be characterized indirectly, for example, using biomarkers and / or directly, through nucleic acid sequencing.

[0080] As used herein, the term "germplasm" refers to genetic material that belongs to or originates from an individual (e.g., a plant), a population of individuals (e.g., a plant strain, variety, or family), or a clone derived from a strain, variety, species, or culture. Germplasm can be part of an organism or cell, or it can be isolated from an organism or cell. Typically, germplasm provides genetic material with a specific molecular structure that provides the physical basis for some or all of the genetic qualities of an organism or cell culture. As used herein, germplasm can refer to seeds, cells (including protoplasts and callus), or tissues from which new plants can grow, as well as plant parts (e.g., stems, buds, roots, leaves, etc.) that can be cultured into a whole plant.

[0081] A "haplotype" is the genotype of an individual at multiple genetic loci, that is, a combination of alleles. Typically, the genetic loci that define a haplotype are physically and genetically linked, that is, located on the same segment of a chromosome. The term "haplotype" can refer to polymorphism at a specific locus (e.g., a single marker locus) or polymorphism at multiple loci along a segment of a chromosome.

[0082] As used in this article, the term "heterozygous" refers to the genetic state in which different alleles are located at corresponding loci on homologous chromosomes.

[0083] As used in this article, the term "homozygous" refers to the genetic state in which the same alleles are located at corresponding loci on homologous chromosomes.

[0084] As used herein, the term "hybrid" refers to the seeds and / or plants produced when at least two genetically dissimilar parents are crossed.

[0085] As used herein, the term "inbred line" refers to a plant or variety that is substantially homozygous. This term can refer to a plant or variety that is substantially homozygous throughout its genome, or to a plant or plant variety that is substantially homozygous relative to a portion of its genome intended for a particular purpose.

[0086] As used herein, the term “indel” refers to an insertion or deletion in a pair of nucleotide sequences, wherein the first sequence may be referred to as having an insertion relative to the second sequence, or the second sequence may be referred to as having a deletion relative to the first sequence.

[0087] As used herein, the terms “introgression,” “introgressing,” and “introgressed” refer to the natural and artificial transfer of a desired allele or combination of desired alleles at one or more genetic loci from one genetic background to another. For example, a desired allele at a designated locus can be transferred to at least one offspring through sexual hybridization between two parents of the same species, wherein at least one of these parents carries the desired allele in its genome. Alternatively, for example, allele transfer can occur through recombination between two donor genomes, such as in fused protoplasts, wherein at least one donor protoplast carries the desired allele in its genome. The desired allele can be a selected allele of a marker, a QTL, a transgene, etc. Offspring containing the desired allele can be repeatedly backcrossed with lines having the desired genetic background, and selection can be performed for the desired allele, wherein the result is that the desired allele becomes fixed in the desired genetic background. For example, biomarkers associated with enhanced ASR tolerance can be introgressed from the donor into recurrent parents that do not have disease resistance. The resulting offspring can then be repeatedly backcrossed and selected until the offspring have one or more ASR tolerance alleles in the recurrent parental background.

[0088] As used herein, the term "expression cassette" refers to a nucleotide capable of directing the expression of a specific nucleic acid sequence in a host cell. In some embodiments, an expression cassette comprises, is substantially composed of, or comprises one or more promoter sequences operatively linked to a target nucleic acid (e.g., one or more constitutive / inducible promoter sequences, one or more tissue-specific and / or organ-specific promoter sequences, and / or one or more developmental stage-specific promoter sequences), the target nucleic acid being operatively linked to a termination sequence. An expression cassette typically contains the sequence required for proper translation of the target nucleic acid sequence in a host cell. An expression cassette may be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components. An expression cassette may be naturally occurring but already obtained in a recombinant form suitable for heterologous expression. However, typically, an expression cassette is heterologous relative to the host (i.e., the specific nucleic acid sequence of the expression cassette is not naturally present in the host cell and must have been introduced into the host cell or its ancestor through a transformation event).

[0089] As used herein, the term "genome editing agent" refers to an agent capable of inducing deletions, insertions, insertion-deletion, or other modifications in the cellular genome, for example, by generating single-strand or double-strand breaks in the genome. Examples of genome editing agents include CRISPR / Cas agents (e.g., Cas proteins and guide RNA), transcription activator-like effector nucleases (TALENs), DNA-directed nucleases, broad-spectrum nucleases, recombinases, and zinc finger nucleases. Cas proteins include Cas9, Cas12a (also known as Cpfl), C2cl, C2c2, and C2c3, and their functional variants. Examples of Cas9 and Cas12a proteins include Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Staphylococcus aureus (SaCas9), Francisella novicida Cas9 (FnCas9), Neisseria cinerea Cas9 (NcCas9), Neisseria meningitis Cas9 (NmCas9), Francisella meningitis Cpfl (FnCpfl), and species of Acidaminococcus. sp.) Cpfl (AsCpfl), or Lachnospiraceae bacteria ND2006 Cpfl (LbCpfl). A “variant” of the Cas protein refers to a protein or polypeptide derivative of the Cas protein, such as a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof. In some embodiments, Cas variants are functional variants that substantially retain the nuclease activity of the wild-type Cas protein or have better nuclease activity than the wild-type Cas protein. Example guide RNAs include single guide RNAs and dual guide RNAs.

[0090] As used herein, the term "heterologous" refers to a polynucleotide / peptide that at least a portion of which is derived from a foreign species, or, if derived from the same species, whose natural form has been substantially modified in terms of composition and / or genomic loci through intentional artificial intervention. Thus, a nucleotide sequence derived from an organism or species different from the one to which the cell to which the nucleotide sequence is introduced is heterologous relative to that cell and its progeny. Additionally, heterologous nucleotide sequences include those derived from and inserted into the same natural progenitor cell type, but existing in a non-natural state (e.g., at a different copy number), and / or under the control of a regulatory sequence different from the regulatory sequence found in the natural state of the nucleic acid molecule. Nucleic acid sequences can also be heterologous to other nucleic acid sequences associated with them, for example, in nucleic acid constructs (e.g., expression vectors). As a non-limiting example, a promoter may exist in a nucleic acid construct in combination with one or more regulatory elements and / or coding sequences that are not naturally associated with that particular promoter (i.e., they are heterologous to that promoter).

[0091] As used herein, when referring to a process or method step, the term "plant in situ" means a process or method step performed on a plant rather than on excised or in vitro cultured plant tissue or organ. For clarity, "plant" includes those that have been traumatized or have had one or more tissues removed, such as plants with traumatized axillary meristems and / or with removed SAMs.

[0092] As used herein, the term "plant in situ transformation" refers to a transformation method performed on a plant without any tissue culture steps on any excised tissue or organ. For clarity, tissue culture steps do not include growing plants on or in growth media, hydroponics, culture plates, etc.

[0093] The terms “nucleic acid” or “polynucleotide” are used interchangeably herein and refer to any physical monomeric unit string that can correspond to a series of nucleotides, including polymers of nucleotides (e.g., typical DNA polymers or polydeoxyribonucleotides or RNA polymers or polynucleotides), modified oligonucleotides (e.g., oligonucleotides containing bases atypical for biological RNA or DNA, such as 2'-O-methylated oligonucleotides), etc. In some embodiments, nucleic acids or polynucleotides may be single-stranded, double-stranded, multi-stranded, or combinations thereof. Unless otherwise stated, in addition to any explicitly indicated polynucleotides, specific nucleic acids or polynucleotides of the present invention optionally also contain or encode complementary polynucleotides. Nucleic acids may be present in vectors, such as cells, viruses, or plasmids.

[0094] As used herein, the phrases “operationally linked,” “operationally linked,” “operationally associated,” or “operationally associated” refer to elements of a nucleic acid construct (such as an expression cassette or nucleic acid molecule) configured to perform their usual function. Therefore, a regulatory or control sequence (e.g., a promoter) that is operationally associated with a nucleotide sequence enables the expression of that nucleotide sequence. For example, a promoter is operationally linked to a coding sequence or functional RNA when it can influence the expression of that coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of that promoter). Coding sequences with positive or antisense orientations can be operationally linked to regulatory sequences. Control sequences do not need to be adjacent to the target nucleotide sequence, as long as they serve to guide its expression.

[0095] Therefore, for example, there may be a non-translated but transcribed sequence between the promoter and the coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0096] The term "plant" refers to any plant, especially those that are agronomically useful (e.g., seed plants), and "plant cell" is the structural and physiological unit of a plant (containing the cell wall), but can also refer to the protoplast. Plant cells can exist as isolated single cells or cultured cells, or as part of higher organized units (e.g., plant tissues, or plant organs that differentiate into structures present at any stage of plant development). Plants can be monocotyledonous or dicotyledonous species.

[0097] The term "plant part" refers to a portion of a plant, including single cells and cellular tissues (e.g., intact plant cells), cell masses, and tissue cultures that can regenerate plants. Examples of plant parts include, but are not limited to, single cells and tissues derived from: pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, buds, and seeds; as well as pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, buds, scions, rootstocks, seeds, protoplasts, callus, etc. The term "plant part" also includes explants.

[0098] The term "offspring" refers to one or more offspring of a particular hybridization. Typically, offspring are produced by breeding two individuals, but some species (particularly some plants and hermaphroditic animals) can self-fertilize (i.e., the same plant acts as a donor for both male and female gametes). One or more offspring can be, for example, F1, F2, or any subsequent generations.

[0099] A “promoter” is a nucleotide sequence that typically lies upstream (5') of its coding sequence and controls its expression by providing recognition of the RNA polymerase and other factors required for appropriate transcription. “Promoter regulatory sequences” consist of proximal and more distal upstream elements. Promoter regulatory sequences influence transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, untranslated leader sequences, introns, and polyadenylation signaling sequences. These include native and synthetic sequences, as well as sequences that may be combinations of synthetic and native sequences. An “enhancer” is a DNA sequence that can stimulate promoter activity, and it can be an intrinsic element of the promoter or a heterologous element inserted to enhance the promoter at a level or tissue specific. It is capable of operating in both orientations (normal or inverted) and can function even when moved upstream or downstream of the promoter. The term “promoter” includes the meaning of “promoter regulatory sequences.”

[0100] As used in this article, the terms “apical meristem”, “tip meristem”, or “SAM” refer to the stem-containing region of a plant located at the tip of its stem.

[0101] In the context of introducing polynucleotides into cells, "stably introducing" or "stably introduced" means that the introduced polynucleotide is stably incorporated into the cell's genome and thus the cell is stably transformed with that polynucleotide.

[0102] As used herein, "stable transformation" or "stable transformation" means the introduction of nucleic acids into a cell and their integration into the cell's genome. Therefore, the integrated nucleic acids can be inherited by their offspring, and more specifically, by offspring across multiple successive generations. As used herein, "genome" also includes the nuclear genome, mitochondrial genome, and plasmid genome, and therefore includes nucleic acids integrated into, for example, the chloroplast genome. As used herein, stable transformation can also refer to transgenes maintained in an extrachromosomal manner (e.g., as mini-chromosomes).

[0103] "Selective agent" refers to a reagent (e.g., a chemical) that interacts with a selectivity marker to give plant cells a selective advantage. Examples of selective agents are known in the art and described herein, such as glyphosate, glufosinate, spectinomycin, bensulfuron-methyl, and kanamycin.

[0104] "Selective markers" or "selective marker genes" refer to genes whose expression in plant cells gives the cells a selective advantage. "Positive selection" refers to the ability of transformed cells to metabolize substrates that were previously unavailable or inefficiently utilized, typically through transformation with and expression of a positive selective marker gene. Such transformed cells then grow from clumps of untransformed tissue. Positive selection can take many forms: from inactive forms of plant growth regulators (which are then converted to active forms by transfer enzymes) to alternative carbohydrate sources (e.g., mannose) that are inefficiently utilized by untransformed cells but become available after conversion with enzymes (e.g., phosphogmannose isomerase), enabling their metabolism. Untransformed cells grow slowly or not at all compared to transformed cells. Other types of selection may result from the ability of cells transformed with selective marker genes to grow in the presence of negative selective agents (e.g., antibiotics or herbicides) compared to the growth capacity of untransformed cells. The selective advantage possessed by transformed cells can also be due to the loss of previously present genes in so-called "negative selection." In this case, the added compound is toxic only to cells that have not lost the specific gene (negative selective marker gene) present in the parent cell (typically a transgene).

[0105] As used herein, the term “transformation” refers to the transfer of nucleic acids into host cells, including integration into chromosomes, heritable extrachromosomal events, and transient transfers. In some specific embodiments, introduction into plants, plant parts, and / or plant cells is carried out via bacterial-mediated transformation. General guidelines for various plant transformation methods known in the art include Miki et al. (“Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, edited by Glick, BR and Thompson, JE (CRC Press, Inc., Boca Raton, 1993), pp. 67–88) and Rakowoczy-Trojanowska (Cell MolBiol Lett 7:849–858 (2002)).

[0106] As used herein, the term "transgenic" means any plant, plant cell, callus, plant tissue, or plant part containing all or part of at least one heteropolynucleotide. In some embodiments, all or part of the heteropolynucleotide is stably integrated into a chromosome or a stable extrachromosomal element such that it is passed on to successive generations.

[0107] Various publications, articles, and patents are cited or described in the background art and throughout the specification; each of these references is incorporated herein by reference in its entirety. All patents, published patent applications, and publications cited herein are incorporated by reference as if fully expounded herein.

[0108] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In other respects, certain terms used herein have the meanings set forth in the specification. Methods of converting sunflowers

[0109] This disclosure provides a method for transforming sunflower seeds using Agrobacterium. The method disclosed provides a highly efficient sunflower transformation system starting from imbibition seeds. This system provides a timeline of only 7 weeks or less to obtain normal and healthy transgenic plants without problems such as premature flowering and rooting. These methods are also independent of germplasm.

[0110] The method disclosed herein can be divided into several distinct stages. Each of these stages may include more than one method step. In some embodiments, these stages may encompass more than one method step. In other embodiments, a single method step may encompass more than one stage.

[0111] like Figure 2A As shown, in some embodiments of this disclosure, these methods include seed germination, co-culture with Agrobacterium containing the target nucleic acid, recovery of transfected sunflower seeds, transplantation of sunflower seeds transfected with the target nucleic acid, and optionally, growth from seed to plant.

[0112] In another embodiment of this disclosure, the methods include: (1) germination of mature sunflower seeds; (2) explant isolation and infection with Agrobacterium containing a target nucleic acid (e.g., heteropolynucleotide); (3) co-culture (contact) in the presence of Agrobacterium containing the target nucleic acid (e.g., heteropolynucleotide); (4) selection of transfected sunflower seeds containing the target nucleic acid; (5) recovery of transfected sunflower seeds; and (6) transplantation into the soil. In some embodiments, the germination step lasts approximately one day. In other embodiments, the co-culture lasts approximately four days. In yet another embodiment, the selection step lasts 7–14 days. In yet another embodiment, the recovery step lasts approximately 3–7 days. In still another embodiment, the transplantation step lasts 2–3 weeks.

[0113] One embodiment of this disclosure relates to a method for directly transforming sunflower seeds from mature seeds using heteropolynucleotides. This method includes at least the following steps: generating explants from germinating sunflower seeds by removing cotyledons and primary leaves containing intact meristems and radicles; gently inducing a wound at the apical meristem region using the sharp tip of a scalpel; optionally contacting the cotyledon-containing sunflower explants with *Agrobacterium tumefaciens* containing heteropolynucleotides in an infection medium supplemented with plant hormones using vacuum and ultrasonic treatment; co-culturing the explants from germinating sunflower plants in a hormone-deficient medium in the presence of *Agrobacterium tumefaciens*; culturing mature seed explants in a regeneration medium containing a selector (e.g., antibiotics such as spectinomycin and bensulfuron-methyl); culturing seeds with regenerated buds, with or without secondary roots, in a selector-deficient medium (e.g., antibiotics such as spectinomycin and bensulfuron-methyl); and transplanting the explants into soil under conditions allowing further development. Explant generation and germination

[0114] The method disclosed herein involves generating explants from germinating sunflower seeds by removing a cotyledon and a primary leaf that have intact meristematic tissue and a radicle.

[0115] In some embodiments, these methods involve germinating sunflower seeds for about a day and then generating explants.

[0116] In some embodiments, the generation of explants involves obtaining hull-less sunflower seeds and sterilizing them. Sterilization can be performed using conventional sterilization methods (such as those described in the examples below).

[0117] In some embodiments, explant generation involves removing at least one cotyledon from a germinating sunflower seed to expose the shoot tip with its two primary leaves. Both primary leaves are completely removed to expose the apical meristem, and the apical region is gently wound. Infection with Agrobacterium spp.

[0118] Once explants are generated (e.g., as described above), they are infected with Agrobacterium (e.g., Agrobacterium tumefaciens containing heteropolynucleotides). In some embodiments, the heteropolynucleotides are genes.

[0119] These methods also include modifying Agrobacterium species (e.g., Agrobacterium tumefaciens) to include heterologous polynucleotides.

[0120] In one embodiment, the method includes contacting sunflower explants containing cotyledons with *Agrobacterium tumefaciens* containing heterologous polynucleotides in an infection medium supplemented with plant hormones, without using vacuum and sonication. In another embodiment, the method includes contacting sunflower explants containing cotyledons with *Agrobacterium tumefaciens* containing heterologous polynucleotides in an infection medium supplemented with plant hormones using vacuum and sonication. In some embodiments, vacuum and sonication improve infection efficiency.

[0121] The method also includes preparing Agrobacterium for infection (i.e., prior to contact). The backup step may include growing Agrobacterium on a suitable culture medium (e.g., YP medium). In one embodiment, preparation of Agrobacterium includes culturing it for approximately three days in a suitable culture medium (e.g., YP medium supplemented with antibiotics) prior to infection (contact). One day before contact, Agrobacterium is cultured in a fresh, suitable culture medium. Prior to infection, Agrobacterium is transferred to a liquid infection medium. In one embodiment, the infection medium contains MS basal salts and vitamin B5, approximately 20 g / L sucrose, approximately 10 g / L glucose, approximately 4 g / L MES, approximately 2 mg / L zeatin nucleoside, approximately 3-10 mg / L TDZ, approximately 80 mg / L acetylsalicylic acid (AS), and approximately 150 mg / L dithiothreitol (DTT), at a pH of approximately 5.5.

[0122] In some embodiments, contacting sunflower explants containing cotyledons with Agrobacterium tumefaciens involves placing the explants in a liquid infection medium containing Agrobacterium. The medium is supplemented with plant hormones. Suitable plant hormones used in the contact step include thidiazuron (1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea) or derivatives thereof, or cytokinins (e.g., 6-benzylaminopurine (BAP)) or derivatives thereof. The medium may be further supplemented with, for example, 10% F68.

[0123] In one embodiment, the infection medium is supplemented with TDZ and / or BAP. In some embodiments, the medium is supplemented with about 3-20 mg / ml, or alternatively about 3-10 mg / ml, or alternatively about 3-15 mg / ml of plant hormones (e.g., TDZ and / or BAP).

[0124] The culture medium containing Agrobacterium and one or more explants are then placed under vacuum and sonicated. In one embodiment, the culture medium containing Agrobacterium and one or more explants are placed under a vacuum of 800-900 mbar for about 5-30 minutes, or alternatively about 5-25 minutes, or alternatively about 20 minutes, and then sonicated at about 45 kHz while gently agitating.

[0125] In some embodiments, contact includes incubation under vacuum and sonication followed by oscillation. Incubation comprises approximately 2-3 hours, and oscillation comprises approximately 50-80 rpm. In some embodiments, sunflowers are contacted in an infection medium containing plant hormones for approximately 4 hours or less.

[0126] In some embodiments of these methods, the contact step with Agrobacterium includes an infection step and an incubation step. In some embodiments of this method, the infection step is performed for at least 30 minutes, for example, from 30 minutes to 24 hours, such as 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, 11-12, 1-11, 2-11, 3-11, 4-11, 5-11, 6-11, 7-11, 8-11, 9-11, 10-11, 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 1-9, 2-9, 3-9, 4-9, 5-9, 6-9, 7-9, 8-9, 1-8, 2-8, 3-8, 4-8, 5-8, 6- 8, 7-8, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 1-6, 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, 4-5, 1-4, 2-4, 3-4, 1-3, 2-3, or 1-2 hours, and the incubation step is carried out in the dark or in the light or in a light / dark cycle for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, or longer, for example, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 1-6, 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, 4-5, 1-4, 2-4, 3-4, 1-3, 2-3, or 1-2 days. In some embodiments, the infection step includes contacting one or more wounded axillary meristems with a solution, gel, absorbable material, or other material containing Agrobacterium. In some embodiments, the infection step lasts 5-12 hours. In some embodiments, the incubation step is performed in the dark for 3-7 days. In some embodiments, after incubation, antibiotics (e.g., termethin, cefotaxime, and / or vancomycin) are applied to eliminate Agrobacterium.

[0127] Agrobacterium-mediated transformation is a method used for transforming plants. Agrobacterium-mediated transformation typically involves the transfer of a binary vector carrying foreign target DNA into a suitable Agrobacterium strain, which may depend on the complementarity of the vir gene carried by the host Agrobacterium strain on a co-existing Ti plasmid or on a chromosome (see, for example, Uknes et al. 1993, Plant Cell 5:159-169). The transfer of the recombinant binary vector into Agrobacterium can be achieved, for example, using a three-parental mating procedure with an Escherichia coli carrying the recombinant binary vector, which is a helper E. coli strain carrying a plasmid capable of moving the recombinant binary vector into the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred into Agrobacterium via nucleic acid transformation (see, for example, Hofgen and Willmitzer 1988, Nucleic Acids Res 16:9877). Co-cultivation

[0128] After contacting sunflower explants containing cotyledons with Agrobacterium tumefaciens in a culture medium supplemented with plant hormones, the resulting explants from germinating sunflower plants were cultured in a culture medium lacking plant hormones in the presence of Agrobacterium tumefaciens.

[0129] In other words, these methods also include co-culturing explants from germinating sunflower plants in the presence of Agrobacterium tumefaciens after removing plant hormones.

[0130] In some embodiments of these methods, co-culture lasts approximately 4-6 days, or alternatively approximately 4 days. In some embodiments, the culture conditions vary during co-culture. For example, in some embodiments, co-culture includes culture with or without light. In some embodiments, co-culture includes culture under low light. In other embodiments, co-culture lasts approximately 3-6 days and involves growing explants in the absence of light.

[0131] In some embodiments, the co-culture step further includes removing the explants from the infection culture medium.

[0132] Co-culture was carried out in a suitable co-culture medium. In some embodiments, the medium was SFCoC, which may be supplemented with MS basal salts and vitamins, MS iron, about 1 mg / L zeatin, about 10 g / L sucrose, about 5 g / L glucose, about 2 g / L LMES, about 20 mg / L acetylsalicylic acid, and about 0.5 mg / ml silver nitrate. regeneration

[0133] like Figure 2B andFigure 2A As shown, after co-culture, transformed sunflower shoots were regenerated. Regeneration involved culturing mature seed explants in a regeneration medium containing a selector, followed by culturing seeds with regenerated shoots and secondary roots in a medium lacking the selector.

[0134] In some embodiments, culturing mature seed explants in a regeneration medium includes inserting cotyledon / meristematic regions into a solid medium in the presence of a selector, covering the seeds with a substrate, and allowing the seeds to grow. In some embodiments, culturing mature seed explants includes culturing for about 7-14 days, alternatively about 8-10 days, alternatively 7-11 days, or alternatively 7-12 days. In alternative embodiments, culturing mature seeds includes culturing in the dark for about the first 2-3 days. In other embodiments, each day of culturing mature seed explants includes culturing for about 16 hours in the presence of light and about 8 hours in the presence of darkness.

[0135] In some embodiments, the culture of mature seed explants includes culturing in a medium supplemented with plant hormones (e.g., BAP). For example, the medium contains SFR1 medium (Gamborg B5 basal salts and B5 vitamins, MS iron 200X, about 20 g / L sucrose, about 1 g / L MES, about 50 mg / L termethin 1, about 150 mg / L carbenicillin, about 7.5 g / L agar, about 50 mM lipoic acid, about 1-4 mg / L BAP and about 50-800 mg / L spectinomycin).

[0136] These methods also include culturing seeds with regenerated shoots, with or without secondary roots, in a medium lacking selectants (such as antibiotics like spectinomycin and bensulfuron-methyl) for a suitable period of time, such as approximately 3–7 days. In some embodiments, the medium lacking selectants is SR1 medium. transplant

[0137] After regeneration (i.e., culturing mature seed explants in a regeneration medium containing a selector, followed by culturing seeds with regenerated shoots in a medium lacking a selector), the explants were transplanted into the soil under conditions that allowed for further development.

[0138] When the explants sprout new shoots with roots, they can be transplanted into the soil.

[0139] In some embodiments, transplanting explants into soil, under conditions that allow for further development, includes allowing the explants to grow for approximately 2–3 weeks without any other options.

[0140] Suitable conditions that allow for further development include, but are not limited to, approximately 27°C for about 20 hours during the day and approximately 20°C for about 4 hours at night.

[0141] In some embodiments, transplantation includes spraying a selector. Therefore, in other embodiments, transplanting explants into the soil to allow for further development includes allowing the explants to grow using a selector. For example, a selector may be sprayed daily after transplantation.

[0142] In one embodiment, the selector is an antibiotic. In other embodiments, the selector is spectinomycin and / or bensulfuron-methyl.

[0143] In some embodiments, these methods include the characterization and / or selection of transgenic plants.

[0144] In some embodiments, selective markers present in the heteropolynucleotide may be used, for example, to select plants, plant parts, and plant cells transformed with heteropolynucleotides using the methods disclosed herein. In some embodiments, one or more selection steps or selectants described in the examples are used to select plants, plant parts, and plant cells transformed with heteropolynucleotides.

[0145] Examples of selective biomarkers include, but are not limited to, providing genes for resistance or tolerance to antibiotics such as kanamycin (Dekeyser et al. 1989, Plant Phys. 90:217-23), spectinomycin (Svab and Maliga 1993, Plant Mol Biol. 14:197-205), streptomycin (Maliga et al. 1988, Mol Gen Genet. 214:456-459), hygromycin B (Waldron et al. 1985, Plant Mol Biol. 5:103-108), bleomycin (Hille et al. 1986, Plant Mol Biol. 7:171-176), and sulfonamides (Guerineau et al. 1990, Plant Mol Biol. 1999). Biol [Plant Molecular Biology] 15:127-136), streptotrichum (Jelenska et al. 2000, Plant Cell Reports 19:298-303) or chloramphenicol (De Block et al. 1984, EMBO J [Journal of the European Society for Molecular Biology] 3:1681-1689). Other selective biomarkers include genes that provide resistance or tolerance to herbicides, such as S4 and / or Hra mutations in acetyl-lactate synthase (ALS) conferring resistance to herbicides (including sulfadiazine, imidazolinone, triazolidine, and thiobenzoates); 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS) genes, including but not limited to those described in U.S. Patent Nos. 4,940,935, 5,188,642, 5,633,435, 6,566,587, 7,674,598 (and all related applications); and glyphosate N-ethylhexyl phosphate synthase. Acyltransferases (GATs) that confer resistance to glyphosate (Castle et al. 2004, Science 304:1151-1154, and U.S. Patent Application Publications 2007 / 0004912, 2005 / 0246798, and 2005 / 0060767); BARs that confer resistance to glufosinate (see, for example, U.S. Patent No. 5,561,236); aryloxyalkyl ester dioxygenases or AAD-1, AAD-12, or AAD-13 that confer resistance to 2,4-D; genes such as those in Pseudomonas HPPD that confer resistance to HPPD.

[0146] Porphyrinone oxidase (PPO) mutants and variants that confer resistance to peroxidative herbicides, including flumetsulam, trifluralin sodium, ethoxyflufenican, quizalofop-p-ethyl, methyl methacrylate, pyrimisulfuron, propyzoxystrobin, fluazinam, pyrazosulfuron, and metsulfuron-methyl; and genes conferring resistance to dicamba (such as dicamba monooxygenase) (Herman et al. 2005, J Biol Chem 280:24759-24767 and U.S. Patent No. 7,812,224, and related applications and patents). Other examples of selective markers can be found in Sundar and Sakthivel (2008, J Plant Physiology 165:1698-1716) (which are incorporated herein by reference). Other selective markers used in this disclosure are known in the art, such as phosphinic acid N-acetyltransferase (PAT) and aminoglycoside 3'-adenylate transferase (ad) (see, for example, Rosellini (2012) Selectable Markers and Reporter Genes: A Well Furnished Toolbox for Plant Science and Genetic Engineering, Critical Reviews in Plant Sciences, 31:5, 401-453).

[0147] Other selection systems include the use of drugs, metabolite analogs, metabolic intermediates, and enzymes for positive or conditional positive selection in transgenic plants. Examples include, but are not limited to, genes encoding phosphomannose isomerase (PMI) (where mannose is the selectant) or genes encoding xylose isomerase (where D-xylose is the selectant) (Haldrup et al. 1998, Plant Mol Biol 37:287-96). Finally, other selection systems can use hormone-free media as the selectant. A non-restrictive example is the corn homeobox gene knl, whose ectopic expression leads to a 3-fold increase in transformation efficiency (Luo et al. 2006, Plant Cell Reports 25:403-409). Examples of various selection markers and their encoding genes are disclosed in Miki and McHugh (J Biotechnol 2004, 107:193-232; incorporated herein by reference).

[0148] In some embodiments disclosed herein, the selective biomarker may be plant-derived. Examples of selective biomarkers that may be plant-derived include, but are not limited to, 5-enolpyruvate-3-phosphate synthase (EPSPS). The enzyme 5-enolpyruvate-3-phosphate synthase (EPSPS) catalyzes an important step in the shikimic acid pathway, a common pathway in the biosynthesis of aromatic amino acids in plants. The herbicide glyphosate inhibits EPSPS, thereby killing the plant. Transgenic glyphosate-tolerant plants, unaffected by glyphosate, can be generated by introducing modified EPSPS transgenes (e.g., U.S. Patent No. 6,040,497; which is incorporated herein by reference). Other examples of modified plant EPSPS that can be used as selective markers in the presence of glyphosate include the P106L mutant of rice EPSPS (Zhou et al. 2006, Plant Physiol 140:184-195) and the P106S mutant of cricket grass EPSPS (Baerson et al. 2002, Plant Physiol 129:1265-1275). Other sources of EPSPS that are not of plant origin and can be used to confer tolerance to glyphosate include, but are not limited to, the P101S mutant of EPSPS from Salmonella typhimurium (Comai et al. 1985, Nature 317:741-744) and a mutant version of CP4 EPSPS from Agrobacterium species strain CP4 (Funke et al. 2006, PNAS 103:13010-13015). Although the plant EPSPS gene is in the cell nucleus, the maturing enzyme is located in the chloroplast (Mousdale and Coggins 1985, Planta 163:241-249). EPSPS is synthesized into a proprotein containing a transport peptide, which is then transported to the chloroplast stroma for protein processing to yield the maturing enzyme (della-Cioppa et al. 1986, PNAS 83:6873-6877). Therefore, to produce transgenic plants tolerant to glyphosate, an appropriate mutant version of EPSPS that is correctly translocated to the chloroplast can be introduced. Such transgenic plants then possess the native genomic EPSPS gene, along with the mutant EPSPS transgene. Glyphosate can then be used as a selector during transformation and regeneration processes, thereby ensuring that only those plants or plant tissues successfully transformed with the mutant EPSPS transgene survive.

[0149] In some embodiments, the selector is an antibiotic, such as spectinomycin and bensulfuron-methyl.

[0150] In some embodiments of the method, the heteropolynucleotide contains a selectivity marker, and the method further includes contacting the plant with a selectivity agent to eliminate or reduce unconverted tissue. In some embodiments, the selectivity agent is a herbicide, antibiotic, or non-metabolizable sugar. In some embodiments, the selectivity agent is glyphosate, glufosinate, spectinomycin, bensulfuron-methyl, metsulfuron-methyl, D-xylose, mannose, or kanamycin. In some embodiments, the selectivity marker is EPSPS, and the selectivity agent is glyphosate.

[0151] In some embodiments of the method, contact with the selector includes adding the selector to a culture medium in which the plant grows (e.g., soil or hydroponics) (e.g., by watering or applying a composition containing the selector, such as a selector between 1 μM and 1 M, for example, 100 μM to 500 μM glyphosate), spraying the plant with the selector (e.g., with a sprayable composition containing the selector, such as a selector between 1 μM and 1 M, for example, 10 μM to 50 mM glyphosate), or applying the selector (e.g., a selector between 1 μM and 1 M, for example, 100 μM to 200 μM glyphosate or 10 μM to 100 μM bensulfuron-methyl) to regenerated axillary meristems (e.g., using a solution, gel, absorbable material (e.g., cotton balls) or other material that releases the selector (e.g., onto wounded axillary meristems and / or regenerated axillary meristems)). In some embodiments, contact with the selective agent lasts for at least one day, at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, or longer. In some embodiments, contact with the selective agent lasts for 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, or 5-6 weeks. In some embodiments, contact with the selective agent lasts for 1 day to 6 weeks. In some embodiments, contact with the selective agent lasts for 3-6 weeks.

[0152] In other embodiments of the method, the method further includes measuring a sample of regenerated axillary meristem to assess the presence and / or number of transformed cells in the sample. Example assays include fluorescent protein detection, qPCR, real-time PCR, immunoassay, etc.

[0153] In alternative embodiments of the method, the method further includes growing a plant to produce seeds (e.g., one seed, two seeds, ten seeds, twenty seeds, fifty seeds, or more) optionally containing at least a portion of a heteropolynucleotide, and harvesting the seeds. In some embodiments, all seeds produced by the plant contain at least a portion of a heteropolynucleotide. In other embodiments, at least one or more seeds produced by the plant (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) contain at least a portion of a heteropolynucleotide. In further embodiments of the method, the method further includes growing one or more seeds to produce one or more progeny plants optionally containing at least a portion of a heteropolynucleotide.

[0154] In another embodiment of the method, the heteropolynucleotide encodes a genome editing agent, such as a CRISPR / Cas reagent, TALEN, DNA-directed nuclease, macronuclease, recombinase, or zinc finger nuclease. In an alternative embodiment of the method, the heteroprotein comprises a genome editing agent, such as a Cas protein, TALEN, DNA-directed nuclease, macronuclease, recombinase, or zinc finger nuclease. In some embodiments, the heteropolynucleotide comprises one or more polynucleotides encoding a Cas protein and / or a guide RNA. In another embodiment, the heteropolynucleotide comprises one or more guide RNAs, optionally wherein the heteropolynucleotide is contained within a ribonucleoprotein (RNP) having a Cas protein. In another embodiment, the Cas protein is Cas9 or Cas12a, or a functional variant thereof.

[0155] In some embodiments of the method, the heteropolynucleotide comprises an expression cassette containing a coding sequence. In other embodiments of the method, the coding sequence encodes a target protein or non-coding RNA. In alternative embodiments, the target protein or non-coding RNA confers one or more desired traits to the plant, such as enhanced growth, increased yield, drought tolerance, salt tolerance, herbicide tolerance, insect resistance, pest resistance, disease resistance, temperature tolerance, enhanced nitrogen use, etc. In some embodiments, the coding sequence encodes a genome editing agent, such as a Cas protein and / or a guide RNA. In some embodiments, the heteropolynucleotide comprises a coding sequence encoding a target protein or non-coding RNA and a coding sequence selectivity marker. In further embodiments of the method, the expression cassette further comprises a promoter operatively linked to one or more coding sequences. The promoter may be, for example, a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0156] Other aspects of this disclosure relate to plants or plant parts produced by any of the methods described above or elsewhere herein (including examples). Other aspects of this disclosure relate to progeny seeds produced by hybridizing a plant produced by any of the methods described above or elsewhere herein with a second plant or by self-pollination of the plant. Other aspects of this disclosure relate to derivatives or commercial products derived from or obtained from plants or plant parts produced by any of the methods described above or elsewhere herein. In some embodiments, commercial products are selected from the group consisting of whole or processed seeds, powders, protein isolates, concentrates, liquids, syrups, pastes, sauces, or other food or products derived from plants or plant parts. Example

[0157] The following examples further illustrate the invention, but do not limit it. Unless otherwise stated, the test materials used in the following examples were purchased from general biochemical reagent stores. For the quantitative tests in the following examples, three replicate tests were set up, and the results were averaged. Example 1: Conventional sunflower conversion process

[0158] Pilot studies of sunflower transformation were conducted using standard in vitro processes typically used for plant transformation. However, these studies failed to generate high-quality transgenic events due to developmental abnormalities during tissue culture, often premature flowering, and rootlessness. Reported conventional sunflower transformation protocols have low transformation frequencies (0.06% to 0.52%), and are often associated with chimeric expression of the introduced genes (Alibert et al. 1999; Muller et al. 2001; Lucas et al. 2000; Hewezi et al. 2001; Molinier et al. 2002; Bidney et al. 1992; Knittel et al. 1994; Malone-Schoneberg et al. 1994; Burrus et al. 1996). Rao and Rohini (1999) used hypocotyls as explants to generate transgenic shoots and produced a 2% transformation frequency, but with rather low rooting efficiency.

[0159] The most efficient regeneration and transformation system using cotyledons as explants was evaluated. After optimizing several transformation parameters (Tables 1, 2, and 3), shoots were generated efficiently (>80% shoot induction rate); however, most shoots were negative, with only about 2% being transgenic (with GFP signal), and positive shoots did not survive due to low rooting rate and growth termination during tissue culture (Table 4). Figure 1A A pilot test of the routine process is shown. Table 1. Bud induction rate of different parts of cotyledons Portion of cotyledon Adaxial Abaxial Cut side Sprout induction rate (%) 42.8 9.4 42.2 Table 2. Shoot induction rate of different plant hormones Early conventional transformation experiments began with mature seeds. Mature seeds produced very low transformation efficiency, and no plant was able to produce viable T1 transgenic seeds, such as... Figure 1B (Rootless, deformed plants) and Figure 1C As shown in the image (early flowering plant). Table 5. Transformations performed using conventional in vitro processes FS5698B achieved a TF of 6.7%, but it had a problem with early flowering and did not produce vigorous T1 seeds. Example 2: Transgenic sunflower produced through Agrobacterium transformation (Program V1)

[0160] Transgenic sunflowers expressing GFP and CFP were generated using variants of the sunflower transformation process. The transformation process used in this example is schematically outlined in... Figure 2A middle. (1) Seed germination

[0161] First, sterilize the seeds. Soak the hulled, dried sunflower seeds in 75% ethanol for 2 minutes. Transfer the seeds to a solution containing 3.5% Clorox and 0.04% silwet-77 for 15 minutes. Then, rinse the seeds with sterile water at least 3 times.

[0162] Sterilized sunflower seeds were transferred to SF Germmedium (Gamborg B5 base salts and vitamins, 20 g / L sucrose, 8 g / L agar, pH 5.6), 20 seeds per dish, and incubated in the dark at 25°C for 20 hours. (2) Preparation of Agrobacterium spp.

[0163] At least 3 days prior to infection, Agrobacterium spp. stored at -80°C were streaked onto YP (yeast peptone) medium containing 100 mg / L spectinomycin and 50 mg / L kanamycin. One day before infection, they were re-streaked onto fresh YP medium plates containing spectinomycin and kanamycin at specific concentrations. Agrobacterium spp. were then collected and resuspended in infection liquid medium (containing MS basal salts and vitamin B5, 20 g / L sucrose, 10 g / L glucose, 4 g / L MES, 2 mg / L zeatin nucleoside, 3-10 mg / L TDZ, 80 mg / L acetylsalicylic acid (AS), 150 mg / L dithiothreitol (DTT), pH 5.5). The concentration of Agrobacterium spp. was adjusted until OD660 = 0.6. (3) Preparation and infection of explants

[0164] Remove one cotyledon from a separated germinating seed with a scalpel to expose the bud tip with its two primary leaves. Remove both primary leaves completely to expose the apical meristem, and gently injure the apical area with the sharp end of a scalpel. See also Figure 3 The resulting explants were then placed in an Agrobacterium suspension with 1 / 1000 10% F68 added; the suspension was then placed under a vacuum of 800-900 mbar for 5-30 min, followed by sonication at 45 kHz for 30-60 s with gentle agitation. The explants were then inoculated with Agrobacterium for 2-3 hours with agitation at 50-80 rpm. (4) Co-cultivation

[0165] After inoculation in (3), the Agrobacterium suspension was removed, and the explants were placed on a filter paper in a culture dish, adaxial side up. The filter paper was pre-wetted with 0.5 ml of co-culture medium (MS basal salts and vitamins, MS iron, zeatin 1 mg / L, sucrose 10 g / L, glucose 5 g / L, MES 2 g / L, acetylsuccinone 20 mg / L, silver nitrate 0.5 mg / L). One side of the filter paper was covered, and the culture dish was sealed with a paraffin film. Approximately 10-20 explants were placed in each culture dish. The explants and Agrobacterium were co-cultured for 4 days under the following low light conditions: PAR (photosynthetically active radiation) of 40 μmol m⁻² s⁻¹, light / dark conditions of 16 / 8. The plates containing the explants were then placed in an incubator ( The explants were covered with steel trays in Scientific Chambers, Perry, Iowa (Perry, IA). These conditions were chosen to increase infection and prevent embryo deterioration. The explants were then co-cultured in the growth chamber in the dark at 22°C for 3–6 days. See Figures 4–5. (5)Regeneration

[0166] After co-culturing, explants were transferred to SFR1 medium (Gamborg B5 basal salts and B5 vitamins, MS iron 200X, sucrose 20 g / L, MES 1 g / L, termethin 150 mg / L, carbenicillin 150 mg / L, agar 7.5 g / L, lipoic acid-50 mM, BAP 1 mg / L) for shoot regeneration. SFR1 medium also contains a selector (i.e., spectinomycin 50-150 mg / L). Explants were allowed to culture in the dark on SFR1 medium at 25°C for 3-6 days, followed by growth in light (16 / 8 light / dark) at 25°C for 9-14 days. Figures 6A-6D The formation of nascent shoots with GFP expression is shown.

[0167] After selection on SFR1 medium with a selector, explants with shoots are transferred to unselected SFR1 medium and allowed to grow for 7–14 days. When the explants produce shoots with complete apical meristems, they can be transplanted into soil. See also Figure 7A and Figure 7B . (6) Transplant the explants into the soil

[0168] Explants with shoot-forming meristems were selected for transplantation into germination trays (soil mixture as substrate) in the GH chamber. Each tray was covered with a plastic dome and placed on seedling racks under the following growing conditions: 27°C for 20 hours during the day and 20°C for 4 hours at night. See Figures 7-8. For ALS transgenic plants, T1 seeds were obtained from the transgenic plants after 3-4 months of growth in the greenhouse. Figure 16. Most transformed sunflower plants had more than 200 seeds, and the seed size and seed plumpness were close to those of wild-type plants. See Figure 16; Table 4. GFP or CFP expression was detected using a fluorescence stereomicroscope. See Figures 18 and 19. 7. Genetic Engineering

[0169] Some T1 seeds of the transgenic plant were sown, and the transgene inheritance was detected using a TaqMan assay. Positive plants with T-DNA were observed in all plants that germinated from the T1 seeds. Therefore, all tested T0 events were capable of transmitting the transgene to the next generation. The results of this test are summarized in Table 5. 8. Reproducibility of the transformation scheme

[0170] The above procedure was repeated, and transgenic plants (events) were obtained repeatedly. These transgenic plants (events) were observed to grow in the greenhouse, and phenotypic analysis showed no abnormalities. See Figure 20. The data in Table 6 clearly demonstrate the reproducibility of the transformation system. 9. Effects of the plant growth regulator TDZ

[0171] Different concentrations of TDZ in the infection medium were tested to assess whether TDZ concentration affected transformation (experimental aliases: SF-52-SP-24545, SF-66-SP-24545, SF-81-SP-24545; variety: F75400). Transformation using explants from seeds that had imbibed for 1 day was performed as described above. TDZ was added to the infection medium during the 2-4 hour infection process. Concentrations of 3-10 mg / L of TDZ were used. TDZ was observed to significantly improve shoot induction rate and transformation efficiency. The results of this test are summarized in Table 7 below. 10. The impact of choosing duration

[0172] The number of days selected was varied to determine whether it had any effect on transformation (experimental references: SF-66-SP-24545, SF-52-SP-24545, SF-54-SP-24545; variety: F75400; vector: 24545).

[0173] For this test, the transformation process using explants from seeds that had imbibed for 1 day was as described above. The results of this test are shown in Table 8 and Figure 21.

[0174] Regeneration on SFR1 medium with the selectant after co-culture was observed to be important. Selection lasted for 4 days without producing transformants. In this experiment, the optimal selection time observed was 9 days, which resulted in 8.2% TF. (Table 8, Figure 21). Therefore, the selection duration with the selectant on the medium should be no less than 4 days.

[0175] These examples illustrate embodiments of the disclosed methods for gene conversion in sunflowers, which are based on Agrobacterium-mediated gene delivery into meristematic cells of fresh imbibition / germination sunflower seeds.

[0176] As these examples demonstrate, multiple transgenic shoot structures were induced from infected explant tissue. The explants were then briefly selected (7–14 days, using spectinomycin as a selector) on a selective medium containing a selector and transplanted into soil to allow shoot and root development under controlled conditions (in a growth chamber). Based on multiple experiments, transgenic events were successfully generated using aadA or ALS as selectivity markers (with spectinomycin and bensulfuron-methyl as selectors, respectively). The transformation frequency (TF) for spectinomycin selection was 8.2%, while the TF for the bensulfuron-methyl selection system was 3.6%.

[0177] The T0 transgenic events generated by this method are normal and healthy, without abnormal phenotypes or developmental problems (e.g., premature flowering, rootlessness), and each T0 event can produce >200 seeds / plant. The timeline for this sunflower transformation method is only about 8 weeks (4 weeks for the in vitro process and 4 weeks for the GH step), significantly shorter than conventional transformation methods that typically take 3 months. Therefore, this disclosure provides a rapid and stable sunflower transformation method for gene manipulation or genome editing. Example 3: Another sunflower conversion scheme (Scheme V2)

[0178] use Figure 2A The scheme shown transforms mature sunflower seeds. The scheme used for transformation is described in detail below. This scheme does not require a germination process (overnight seed imbibition) and instead relies on the direct transformation of mature seeds. The scheme has the following six general steps. (1) Seed germination

[0179] Allow the seeds to absorb water for 4-48 hours, ideally about 20 hours before separating the explants. (2) Preparation of Agrobacterium spp.

[0180] At least 3 days prior to infection, Agrobacterium spp. stored at -80°C were streaked onto YP (yeast peptone) medium containing 100 mg / L spectinomycin and 50 mg / L kanamycin. One day before infection, they were re-streaked onto fresh YP medium plates containing spectinomycin and kanamycin at specific concentrations. Agrobacterium spp. were then collected and resuspended in infection liquid medium (containing MS basal salts and vitamin B5, 20 g / L sucrose, 10 g / L glucose, 4 g / L MES, 2 mg / L zeatin nucleoside, 3-10 mg / L TDZ, 80 mg / L acetylsalicylic acid (AS), 150 mg / L dithiothreitol (DTT), pH 5.5). The concentration of Agrobacterium spp. was adjusted until OD660 = 0.6. (3) Preparation and infection of explants

[0181] To isolate the explants, the imbibed seeds are separated, one cotyledon is removed, the primary leaf is removed, a wound is created between the apical regions, and the entire hypocotyl is preserved, thus generating the explants. This method does not require the use of induced / existing axillary buds / apical meristems before inoculation. The primary meristems are physically destroyed prior to inoculation (infection). The resulting explants are infected with Agrobacterium by culturing the explants in the presence of 3-20 mg / L thiamethoxam (TDZ) in the infection medium. In particular, to minimize the negative effects of prolonged presence of TDZ during tissue culture, a higher concentration of TDZ (3-20 mg / L) in our Agrobacterium suspension is used only during inoculation (<4 hours), after which it is removed to induce shoots during co-culture and subsequent tissue culture steps. Infection involves sonication at 45 kHz for one minute followed by 30 minutes under vacuum. (4) Co-cultivation

[0182] After inoculation, the Agrobacterium suspension was removed, and the explants were placed on a piece of filter paper in a culture dish, adaxial side up. The filter paper was pre-moistened with 0.5 ml of co-culture medium (MS basal salts and vitamins, MS iron, zeatin 1 mg / L, sucrose 10 g / L, glucose 5 g / L, MES 2 g / L, acetylsuccione 20 mg / L, silver nitrate 0.5 mg / L). One side of the filter paper was covered, and the culture dish was sealed with paraffin film. Approximately 10-20 explants were placed in each culture dish. The explants and Agrobacterium were co-cultured for 4 days under the following low-light conditions: PAR (photosynthetically active radiation) of 40 μmol / L. -2 s -1 The light / dark ratio was 16 / 8. We placed the plates containing the explants in an incubator ( The incubator, located at Scientific Chambers, Perry, Iowa, was covered with a steel tray and set to 22°C. The optical module was from SciWhite. TM LED light bricks. (5)Regeneration

[0183] Transformed sunflower explants were transferred to regeneration medium by inserting cotyledons with meristem regions into a solid medium containing a selection agent. The hypocotyl was kept out of contact with the medium. The hypocotyl was covered with sterile substrate, and the container was placed at 22°C under 16 / 8h light / dark conditions. The regeneration process lasted 7–14 days. Specifically, after co-culture, mature seeds were placed in regeneration medium as follows: cotyledons / merem regions were inserted into a solid medium containing a selection agent (100 mg / L spectinomycin), with the hypocotyl facing upwards (the entire seed inverted); the container was covered with sterile substrate (soil mixture) and placed in a growth chamber to simultaneously induce shoots and roots under short-term (7–14 days) selection. (6) Rooting

[0184] Subsequently, the transformed explants were transferred to rooting media for root induction and development. Specifically, the explants were removed from the regeneration medium and then placed in a rooting medium lacking selectants. The explants were allowed to grow in the rooting medium for 3–14 days until shoots and roots were formed. (7) Transplant the explants into the soil

[0185] After shoots and roots have developed, the plants are transplanted into soil and covered with a plastic dome to maintain humidity and light, allowing them to grow for 2–4 weeks. Only plants with good shoots and roots are transplanted into soil and grown in a greenhouse without any selection. These plants are then characterized. 1. Improved rooting rate from method version 1 (described in example 2) to method version 2 (described in example 3). Overall, rooted plantlets were more likely to survive in the greenhouse than rootless plantlets. The rooting rate increased from an average of <10% to >75%, significantly improving the survival rate of plants after transplanting into soil. This approach was also applied to another variety, as demonstrated in cultivar FS5698B, achieving a rooting frequency of 88%. 2. Scheme V2 improves the conversion frequency compared to Scheme V1. Table 10 shows the results of the conversion experiments conducted using F75400, comparing the conversion frequencies of conversion scheme version 1 and version 2 side by side, and observing a significant improvement in TF. 3. Reproducibility of the rapid conversion scheme for the improved method To test the reproducibility of the improved transformation protocol V2, variety F75400 and construct 24545 were used. Positive events were regenerated using normal shoots and roots and further confirmed by molecular analysis. Table 11 below shows the reproducibility of the transformation protocol and the duration of the entire process (ranging from 41 to 56 days), demonstrating the speed and reliability of the new protocol. 4. Cytokinin preconditioning (e.g., TDZ, but not limited to TDZ) Adding different cytokinins to the germination medium improved shoot induction rate and TF. Resistant plantlets with normal shoots and roots were generated, which was further confirmed by visualization of green fluorescent protein (GFP) expression. (Experimental aliases: SF-52-SP-24545, SF-66-SP-24545, SF-81-SP-24545; Variety: F75400). 5. Duration of seed germination: Seeds that have swelled in the culture medium for 4-48 hours can increase the frequency of spectinomycin-resistant shoot induction. (Experimental aliases: SF-55-SP-24545, variety: F70219; SF-23-SP-24545, SF-22-SP-24545, variety: F75400). 6. Select the range of spectinomycin concentration in the culture medium (spectinomycin 100 mg / L - 800 mg / L); The test was conducted on selective media containing 100–800 mg / L spectinomycin. Resistant plantlets with normal shoots and roots were generated, further confirmed by GFP visualization. All treatments with spectinomycin concentrations ranging from 100 mg / L to 800 mg / L generated resistant shoots. (Experimental alias: SF-23-SP-24545; Variety: F75400) 7. Genotype independence The flexibility of the transformation scheme was evaluated using the V2 method in several superior sunflower lines. Positive events generated exhibited normal shoots and roots, confirmed by GFP visualization. Data showed that, in addition to the model line F75400 and another line F70219, nine other superior lines were available for transformation using the V2 method.

[0186] It should be understood that although this disclosure has been described in conjunction with preferred specific embodiments, the foregoing description and the following examples are intended to illustrate and not limit the scope of this disclosure. Those skilled in the art will understand that various changes can be made and equivalents can be substituted without departing from the scope of this disclosure; and other aspects, advantages, and modifications will be apparent to those skilled in the art to which this disclosure pertains. In addition to the embodiments described herein, this disclosure also covers and claims inventions resulting from combinations of features of this disclosure cited herein and features of prior art references cited to supplement this disclosure. Similarly, it will be understood that any described material, feature, or article of manufacture may be used in combination with any other material, feature, or article of manufacture, and such combinations are considered to be within the scope of this disclosure.

Claims

1. A method for directly converting sunflower plants from mature seeds using heterologous polynucleotides, the method comprising: a) Explants are generated from germinating sunflower seeds by separating cotyledons and primary leaves with complete meristematic tissue and radicles. b) Contact the sunflower explant containing the cotyledons with infecting bacteria containing heterologous polynucleotides in an infection medium supplemented with plant hormones; c) The explants from germinating sunflower plants are co-cultured in a culture medium lacking the plant hormones in the presence of the infecting bacteria; d) The mature seed explants were cultured in a regeneration medium containing a selector; e) Cultivating the seeds with regenerated shoots and secondary roots in a medium lacking a selector; and f) Transplant the explants into the soil, provided that further development is allowed.

2. The method of claim 1, wherein the method further comprises germinating sunflower seeds for approximately one day.

3. The method of claim 1, wherein the infecting bacteria comprises Agrobacterium tumefaciens.

4. The method of any one of claims 1-3, wherein the infecting bacteria is modified to contain the heteropolynucleotide.

5. The method of claim 4, wherein the infectious bacteria are modified by transformation.

6. The method of any one of claims 1-5, wherein the heteropolynucleotide is a gene.

7. The method of any one of claims 1-6, wherein vacuum and ultrasonic treatment are used to bring the sunflower explant into contact with the infecting bacteria.

8. The method of claim 7, wherein the vacuum and ultrasonic treatment improve infection efficiency.

9. The method according to any one of claims 1-8, wherein the plant hormone comprises one or more of TDZ, BAP, zeatin, and kinetin.

10. The method of claim 9, wherein the concentration of the plant hormone is about 3-20 mg / ml.

11. The method of any one of claims 1-10, wherein the method comprises contacting the sunflower with an infection culture medium containing the plant hormone for about 4 hours or less.

12. The method of any one of claims 1-11, wherein the co-culture comprises culturing with or without light.

13. The method of claim 12, wherein the co-culture comprises culturing under low light conditions.

14. The method of claim 12 or 13, wherein the method comprises co-culturing for approximately 4-6 days.

15. The method of any one of claims 1-14, wherein culturing the mature seed explants in a regeneration medium comprises inserting the cotyledon / meriste region into a solid culture medium in the presence of a selector, covering the seed with a substrate, and allowing the seed to grow.

16. The method of claim 15, wherein the method includes a culture period of approximately 7-14 days.

17. The method of claim 16, wherein the method comprises culturing in the dark for about 2-3 days prior.

18. The method of claim 15, wherein the 24-hour incubation comprises 16 hours of incubation in the presence of light and 8 hours of incubation in the presence of darkness.

19. The method of any one of claims 1-18, wherein culturing the seeds having regenerated shoots and secondary roots in a culture medium lacking a selector comprises a culturing period of approximately 3-7 days.

20. The method of any one of claims 1-19, wherein transplanting the explant into the soil under conditions allowing further development comprises allowing the explant to grow for about 2-3 weeks without a selector.

21. The method of any one of claims 1-19, wherein transplanting the explant into the soil under conditions allowing further development includes allowing the explant to grow in the presence of a selection agent.

22. The method of any one of claims 1-20, wherein the method further comprises characterizing the transgenic plant.

23. The method of claim 21, further comprising determining the presence of the heteropolynucleotide in the transgenic plant.

24. The method of any one of claims 1-23, wherein the selector comprises an antibiotic.

25. The method of claim 24, wherein the selector comprises spectinomycin, bensulfuron-methyl, or one or more combinations thereof.

26. A plant or plant part, said plant or plant part being produced by the method of any one of claims 1-25.

27. A progeny seed, said progeny seed being produced by: a) hybridizing the plant of claim 26 with a second plant, or b) self-pollinating the plant of claim 26.

28. A derivative or product derived from or obtained from the plant or plant part as described in claim 26.

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